This invention relates to antifungal proteins, processes for their manufacture and use, and DNA sequences encoding them.
In this context, antifungal proteins are defined as proteins or peptides possessing antifungal activity. Activity includes a range of antagonistic effects such as partial inhibition or death.
A wide range of antifungal proteins with activity against plant pathogenic fungi have been isolated from certain plant species. We have previously described a class of antifungal proteins capable of isolation from radish and other plant species. These proteins are described in the following publications which are specifically incorporated herein by reference: International Patent Application Publication Number WO93/05153 published Mar. 18, 1993; Terras FRG et al, 1992, J Biol Chem, 267:15301-15309; Terras et al, FEBS Lett, 1993, 316:233-240; Terras et al, 1995, Plant Cell, 7:573-588. The class includes Rs-AFP1 (antifungal protein 1), Rs-AFP2, Rs-AFP3 and Rs-AFP4 from Raphanus sativus and homologous proteins such as Bn-AFP1 and Bn-AFP2 from Brassica napus, Br-AFP1 and Br-AFP2 from Brassica rapa, Sa-AFP1 and Sa-AFP2 from Sinapis alba, At-AFP1 from Arabidopsis thaliana, Dm-AMP1 and Dm-AMP2 from Dahlia merckii, Cb-AMP1 and Cb-AMP2 from Cnicus benedictus, Lc-AFP from Lathyrus cicera, Ct-AMP1 and Ct-AMP2 from Clitoria ternatea. The proteins specifically inhibit a range of fungi and may be used as fungicides for agricultural or pharmaceutical or preservative purposes. It has been proposed that this class of antifungal proteins should be named as plant defensins (Terras F. R. G. et al 1995, Plant Cell 7 573-588) and these proteins share a similar motif of conserved cysteines and glycines (Broekaert et al 1995 Plant Physiol 108 1353-1358).
FIG. 1 shows the amino acid sequences of the protein Rs-AFP2 (SEQ ID NO: 9) and the substantially homologous proteins Rs-AFP1 (SEQ ID NO: 8), Rs-AFP3 (SEQ ID NO: 10), Rs-AFP4 (SEQ ID NO: 11), Br-AFP1 (SEQ ID NO: 12), Br-AFP2 (SEQ ID NO: 13), Bn-AFP1 (SEQ ID NO: 14), Bn-AFP2 (SEQ ID NO: 15), Sa-AFP1 (SEQ ID NO: 16), Sa-AFP2 (SEQ ID NO: 17) and At-AFP1 (SEQ ID NO: 18) which are small 5 kDa polypeptides that are highly basic and rich in cysteine. FIG. 1 numbers the positions of the amino acid residues: the dash (-) at the start of the Rs-AFP3 sequence indicates a gap introduced for maximum alignment. The sequences shown for Br-AFP1, Br-AFP2, Bn-AFP1, Bn-AFP2, Sa-AFP1, Sa-AFP2 and At-AFP1 are not complete: only the N-terminal sequences are shown. The question mark (?) in the Br-AFP2 sequence indicates a non-standard amino acid which the sequencing could not assign and which is thought to be a post-translational modification on one of the standard amino acid residues.
The primary structures of the two antifungal protein isoforms capable of isolation from radish seeds, Rs-AFP1 (SEQ ID NO: 8) and Rs-AFP2 (SEQ ID NO: 9), only differ at two positions: the glutamic acid residue (E) at position 5 in Rs-AFP1 (SEQ ID NO: 8) is a glutamine residue (Q) in Rs-AFP2 (SEQ ID NO: 9), and the asparagine residue (N) at position 27 in Rs-AFP1 (SEQ ID NO: 8) is substituted by an arginine residue (R) in Rs-AFP2 (SEQ ID NO: 9). As a result, Rs-AFP2 (SEQ ID NO: 9) has a higher net positive charge (+2) at physiological pH. Although both Rs-AFPs are 94% identical at the amino acid sequence level, Rs-AFP2 (SEQ ID NO: 9) is two- to thirty-fold more active than Rs-AFP1 (SEQ ID NO: 8) on various fungi and shows an increased salt-tolerence. The proteins Rs-AFP3 (SEQ ID NO: 10) and Rs-AFP4 (SEQ ID NO: 11) are found in radish leaves following localized fungal infection. The induced leaf proteins are homologous to Rs-AFP1 (SEQ ID NO: 8) and Rs-AFP2 (SEQ ID NO: 9) and exert similar antifungal activity in vitro.
The cDNA encoding Rs-AFP1 (SEQ ID NO: 19) encodes a preprotein with a signal peptide followed by the mature protein. The cDNA sequence is shown n FIG. 2. Saccharomyces cerevisiae can be used as a vector for the production and secretion of Rs-AFP2 (Vilas Alves et al, FEBS Lett, 1994, 348:228-232). Plant-derivable xe2x80x9cwild-typexe2x80x9d Rs-AFP2 can be correctly processed and secreted by yeast when expressed as a N-terminal fusion to the yeast mating factor xcex11 (MFxcex11) preprosequence. The Rs-AFP2 protein does not have adverse effects on yeast even at concentrations as high as 500 xcexcg/ml.
We now provide new potent antifungal proteins based on the structure of the Rs-AFPs and related proteins.
According to a first aspect the invention provides an antifungal protein having an amino acid sequence which is substantially homologous to the Rs-AFP2 sequence (SEQ ID NO: 9) shown in FIG. 1 and containing at least one mutation selected from the group consisting of a basic residue at position 9, a basic residue at position 39, a hydrophobic residue at position 5 and a hydrophobic residue at position 16.
According to a preferred embodiment of the first aspect of the present invention there is provided an antifungal protein having an amino acid sequence which is substantially homologous to the Rs-AFP2 sequence (SEQ ID NO: 9) shown in FIG. 1 and containing at least one mutation selected from the group consisting of an arginine residue at position 9, an arginine residue at position 39, a methionine residue at position 5 and a methionine residue at position 16. An antifungal protein having both a mutation to arginine at position 9 and a mutation to arginine at position 39 may be particularly active.
Proteins which are substantially homologous to the Rs-AFP2 protein include the proteins Rs-AFP1 (SEQ ID NO: 8), Rs-AFP3 (SEQ ID NO: 10), Rs-AFP4 (SEQ ID NO: 11), Br-AFP1 (SEQ ID NO: 12), Br-AFP2 (SEQ ID NO: 13), Bn-AFP1 (SEQ ID NO: 14), Bn-AFP2 (SEQ ID NO: 15), Sa-AFP1 (SEQ ID NO: 16), Sa-AFP2 (SEQ ID NO: 17) and At-AFP1 (SEQ ID NO: 18) shown in FIG. 1.
As used herein the term substantially homologous denotes those proteins which have an amino acid sequence with at least 40% identity, preferably at least 60% identity and most preferably at least 80% identity to the Rs-AFP2 sequence (SEQ ID NO 9).
The invention further provides an antifungal peptide which comprises at least six amino acid residues identical to a run of amino acid residues in an antifungal protein according to the invention, said run of residues including at least one of the mutated residues.
In particular, there are provided the following antifungal proteins and antifungal peptides derived therefrom:
a protein having the amino acid sequence of Rs-AFP1 (SEQ ID NO: 8), Rs-AFP2 (SEQ ID NO: 9), Rs-AFP3 (SEQ ID NO: 10) or Rs-AFP4 (SEQ ID NO: 11) in which the glycine residue at position 9 is replaced by an arginine residue;
a protein having the amino acid sequence of Rs-AFP1 (SEQ ID NO: 8), Rs-AFP2 (SEQ ID NO: 9) or Rs-AFP3 (SEQ ID NO: 10) in which the valine residue at position 39 is replaced by an arginine residue;
a protein having the amino acid sequence of Rs-AFP4 (SEQ ID NO: 11) in which the isoleucine residue at position 39 is replaced by an arginine residue;
a protein having the amino acid sequence of Rs-APF1 (SEQ ID NO: 8), Rs-AFP2 (SEQ ID NO: 9) or Rs-AFP3 (SEQ ID NO: 10) in which the glycine residue at position 9 is replaced by an arginine residue and the valine residue at position 39 is replaced by an arginine residue;
a protein having the amino acid sequence of Rs-AFP4 (SEQ ID NO: 11) in which the glycine residue at position 9 is replaced by an arginine residue and the isoleucine residue at position 39 is replaced by an arginine residue;
a protein having the amino acid sequence of Rs-AFP1 (SEQ ID NO: 8), Rs-AFP3 (SEQ ID NO: 10) or Rs-AFP4 (SEQ ID NO: 11) in which the glutamic acid residue at position 5 is replaced by a methionine residue;
a protein having the amino acid sequence of Rs-AFP2 (SEQ ID NO: 9)in which the glutamine residue at position 5 is replaced by a methionine residue;
a protein having the amino acid sequence of Rs-AFP1 (SEQ ID NO: 8), RS-AFP2 (SEQ ID NO: 9), Rs-AFP3 (SEQ ID NO: 10) or Rs-AFP4 (SEQ ID NO: 11) in which the glycine residue at position 16 is replaced by a methionine residue.
Proteins according to the invention include proteins having one of the following sequences:
QKLCERPSRTWSGVCGNNNACKNQCINLEKARHGSCNYVFPAHKCICYFPC (SEQ ID NO: 57);
QKLCERPSGTWSGVCGNNNACKNQCINLEKARHGSCNYRFPAHKCICYFPC (SEQ ID NO: 58);
QKLCERPSRTWSGVCGNNNACKNQCINLEKARHGSCNYRFPAHKCICYFPC (SEQ ID NO: 59);
QKLCMRPSGTWSGVCGNNNACKNQCINLEKARHGSCNYVFPAHKCICYFPC (SEQ ID NO: 60);
QKLCERPSGTWSGVCMNNNACKNQCINLEKARHGSCNYVFPAHKCICYFPC (SEQ ID NO: 61);
QKLCQRPSRTWSGVCGNNNACKNQCIRLEKARHGSCNYVFPAHKCICYFPC (SEQ ID NO: 62);
QKLCQRPSGTWSGVCGNNNACKNQCIRLEKARHGSCNYRFPAHKCICYFPC (SEQ ID NO: 63);
QKLCQRPSRTWSGVCGNNNACKNQCIRLEKARHGSCNYRFPAHKCICYFPC (SEQ ID NO: 64);
QKLCMRPSGTWSGVCGNNNACKNQCIRLEKARHGSCNYVFPAHKCICYFPC (SEQ ID NO: 65);
QKLCQRPSGTWSGVCMNNNACKNQCIRLEKARHGSCNYVFPAHKCICYFPC (SEQ ID NO: 66);
KLCERSSRTWSGVCGNNNACKNQCIRLEGAQHGSCNYVFPAHKCICYFPC (SEQ ID NO: 67);
KLCERSSGTWSGVCGNNNACKNQCIRLEGAQHGSCNYRFPAHKCICYFPC (SEQ ID NO: 68);
KLCERSSRTWSGVCGNNNACKNQCIRLEGAQHGSCNYRFPAHKCICYFPC (SEQ ID NO: 69);
KLCMRSSGTWSGVCGNNNACKNQCIRLEGAQHGSCNYVFPAHIKCICYFPC (SEQ ID NO: 70);
KLCERSSGTWSGVCMNNNACKNQCIRLEGAQHGSCNYVFPAHKCICYFPC (SEQ ID NO: 71);
QKLCERSSRTWSGVCGNNNACKNQCINLEGARHGSCNYFPYHRCICYFPC (SEQ ID NO: 72);
QKLCERSSGTWSGVCGNNNACKNQCINLEGARHGSCNYRFPYHRCICYFPC (SEQ ID NO: 73);
QKLCERSSRFWSGVCGNNNACKNQCINLEGARHGSCNYRFPYHRCICYFPC (SEQ ID NO: 74);
QKLCMRSSGTWSGVCGNNNACKNQCINLEGARHGSCNYIFPYHRCICYFPC (SEQ ID NO: 75);
QKLCERSSGTWSGVCMNNNACKNQCINLEGARHGSCNYIFPYHRCICYFPC (SEQ ID NO: 76).
A cDNA clone encoding the plant-derivable xe2x80x9cwild-typexe2x80x9d Rs-AFP2 preprotein was modified by recombinant DNA methods in order to allow expression in the yeast Saccharomyces cerevisiae. This peptide was expressed in yeast as a fusion protein carrying at its N-terminus the prepro sequences derived from the precursor of the yeast pheromone mating factor xcex11. These sequences allow secretion of the biologically active peptide in a correctly processed form. The yeast expression system was then used to express and characterize isoforms of the Rs-AFP2 protein by introducing deliberate or random changes into the coding region. These isoforms were subsequently purified and tested for their antifungal activity.
The Rs-AFP2 isoform having a mutation at position 5 (glutamine to methionine) (SEQ ID NO: 22) and the Rs-AFP2 isoform having a mutation at position 16 (glycine to methionine) (SEQ ID NO: 25) have an enhanced salt-tolerant antifungal activity. However, two other isoforms were found to possess particularly advantageous antifungal properties. The Rs-AFP2 isoform having a mutation at position 9 (glycine to arginine) (SEQ ID NO: 38) and the Rs-AFP2 isoform having a mutation at position 39 (valine to arginine) (SEQ ID NO: 43) have a significantly enhanced antifungal activity. This enhanced activity is prominent in high salt conditions. An Rs-AFP2 isoform having a mutation at both position 9 (glycine to arginine) and at position 39 (valine to arginine) may have an even greater salt-tolerance.
Proteins which maintain their antifungal activity as salt concentration is increased are particularly suitable for use as antifungal agents in higher salt conditions. For example, such proteins are particularly suitable for expression within some biological organisms including plants. The most abundant divalent cations in plant tissues are Ca2+ and Mg2+. The concentration of free Ca2+ in the cytosol is very low (0.1 to 1 xcexcM) (Macklom, 1984, Plant Cell Environ. 7:407-413)), whereas free Mg2+ reaches about 1 mM (Hepler and Wyne, 1982, Ann Rev Plant Physiol, 36:397-439). Free Ca2+ in plant vacuoles is about 0.06 to 1 mM and apoplastic free Ca2+ ranges between 0.02 and 1.3 mM (Harker and Venis, 1991, Plant Cell Environ, 14:525-530). It thus appears that relatively high ionic strength conditions occur in all cellular compartments. In many cases, however, fungal infection leads to the disruption of the cells and contact of the cellular contents with the environment. Therefore it is difficult to predict the exact ionic conditions under which antifungal proteins expressed within a plant cell will interact with invading hyphae. However, proteins whose antifungal activity is less sensitive to cation concentration are particularly suitable for expression within plant cells.
An antifungal protein according to the invention may be manufactured from its known amino acid sequence by chemical synthesis using a standard peptide synthesiser, or produced within a suitable organism (for example, a micro-organism or plant) by expression of recombinant DNA. The antifungal protein is useful as a fungicide and may be used for agricultural or pharmaceutical applications.
Knowledge of its primary structure enables manufacture of the antifungal protein, or parts thereof, by chemical synthesis using a standard peptide synthesiser. It also enables production of DNA constructs encoding the antifungal protein.
The invention further provides a DNA sequence encoding an antifungal protein according to the invention. The DNA sequence may be predicted from the known amino acid sequence and DNA encoding the protein may be manufactured using a standard nucleic acid synthesiser. Alternatively, DNA encoding proteins according to the invention may be produced by appropriate site-directed mutagenesis of DNA sequences encoding one of the proteins shown in FIG. 1.
The DNA sequence encoding the antifungal protein may be incorporated into a DNA construct or vector in combination with suitable regulatory sequences (promoter, terminator, transit peptide etc). The DNA sequence may be placed under the control of a homologous or heterologous promoter which may be a constitutive or an inducible promoter (stimulated by, for example, environmental conditions, presence of a pathogen, presence of a chemical). The transit peptide may be a homologous or heterologous to the antifungal protein and will be chosen to ensure secretion to the desired organelle or to the extracellular space. The transit peptide is preferably that naturally associated with the antifungal protein of interest.
Such a DNA construct may be cloned or transformed into a biological system which allows expression of the encoded protein or an active part of the protein. Suitable biological systems include micro-organisms (for example, bacteria such as Escherichia coli, Pseudomonas and endophytes such as Clavibacter xyli subsp. cynodontis (Cxc); yeast; viruses; bacteriophages; etc), cultured cells (such as insect cells, mammalian cells) and plants. In some cases, the expressed protein may subsequently be extracted and isolated for use.
An antifungal protein according to the invention is useful for combatting fungal diseases in plants. The invention further provides a process of combating fungi whereby they are exposed to an antifungal protein according to the invention.
For pharmaceutical applications, the antifungal protein may be used as a fungicide to treat mammalian infections (for example, to combat yeasts such as Candida).
An antifungal protein according to the invention may also be used as a preservative (for example, as a food additive).
For agricultural applications the antifungal protein may be used to improve the disease-resistance or disease-tolerance of crops either during the life of the plant or for post-harvest crop protection. Pathogens exposed to the proteins are inhibited. The antifungal protein may eradicate a pathogen already established on the plant or may protect the plant from future pathogen attack. The eradicant effect of the protein is particularly advantageous.
Exposure of a plant pathogen to an antifungal protein may be achieved in various ways, for example:
(a) The isolated protein may be applied to plant parts or to the soil or other growth medium surrounding the roots of the plants or to the seed of the plant before it is sown using standard agricultural techniques (such as spraying).
The protein may have been extracted from plant tissue or chemically synthesised or extracted from microorganisms genetically modified to express the protein. The protein may be applied to plants or to the plant growth medium in the form of a composition comprising the protein in admixture with a solid or liquid diluent and optionally various adjuvants such as surface-active agents. Solid compositions may be in the form of dispersible powders, granules, or grains.
(b) A composition comprising a micro-organism genetically modified to express the antifungal protein may be applied to a plant or the soil in which a plant grows.
(c) An endophyte genetically modified to express the antifungal protein may be introduced into the plant tissue (for example, via a seed treatment process).
An endophyte is defined as a micro-organism having the ability to enter into non-pathogenic endosymbiotic relationships with a plant host. A method of endophyte-enhanced protection of plants has been described in a series of patent applications by Crop Genetics International Corporation (for example, International Application Publication Number WO90/13224 European Patent Publication Number EP-125468-B1, International Application Publication Number WO91/10363, International Application Publication Number WO87/03303). The endophyte may be genetically modified to produce agricultural chemicals. International Patent Application Publication Number WO94/16076 (ZENECA Limited) describes the use of endophytes which have been genetically modified to express a plant-derived antifungal protein.
(d) DNA encoding an antifungal protein may be introduced into the plant genome so that the protein is expressed within the plant body (the DNA may be cDNA, genomic DNA or DNA manufactured using a standard nucleic acid synthesiser).
Plant cells may be transformed with recombinant DNA constructs according to a variety of known methods (Agrobacterium Ti plasmids, electroporation, microinjection, microprojectile gun, etc). The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocotyledonous and dicotyledonous plants may be obtained in this way, although the latter are usually more easy to regenerate. Some of the progeny of these primary transformants will inherit the recombinant DNA encoding the antifungal protein(s).
The invention further provides a plant having improved resistance to a fungal pathogen and containing recombinant DNA which expresses an antifungal protein according to the invention. Such a plant may be used as a parent in standard plant breeding crosses to develop hybrids and lines having improved fungal resistance.
Recombinant DNA is DNA, preferably heterologous, which has been introduced into the plant or its ancestors by transformation. The recombinant DNA encodes an antifungal protein expressed for delivery to a site of pathogen attack (such as the leaves). The DNA may encode an active subunit of an antifungal protein.
A pathogen may be any fungus growing on, in or near the plant. In this context, improved resistance is defined as enhanced tolerance to a fungal pathogen when compared to a wild-type plant. Resistance may vary from a slight increase in tolerance to the effects of the pathogen (where the pathogen in partially inhibited) to total resistance so that the plant is unaffected by the presence of pathogen (where the pathogen is severely inhibited or killed). An increased level of resistance against a particular pathogen or resistance against a wider spectrum of pathogens may both constitute an improvement in resistance. Transgenic plants (or plants derived therefrom) showing improved resistance are selected following plant transformation or subsequent crossing.
Where the antifungal protein is expressed within a transgenic plant or its progeny, the fungus is exposed to the protein at the site of pathogen attack on the plant. In particular, by use of appropriate gene regulatory sequences, the protein may be produced in vivo when and where it will be most effective. For example, the protein may be produced within parts of the plant where it is not normally expressed in quantity but where disease resistance is important (such as in the leaves).
Examples of genetically modified plants which may be produced include field crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, onion.